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Systems and methods for combined detection of genetic alterations

a combined detection and gene technology, applied in the field of precision medicine, can solve the problems of loss of both, cost and time-consuming separation process, etc., and achieve the effect of reducing sequencing and pcr errors and increasing barcode diversity

Active Publication Date: 2021-11-16
PREDICINE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0023]The present invention further disclosed that Gene RADAR (RNA and DNA single molecular digital Reading) bioinformatics analysis tool can decipher two-layer RNA molecular barcoding to: (1) enable differentiation of the RNA derived reads from DNA derived reads by checking the RNA specific tags in the sequence reads; (2) enable the suppression of the sequencing and PCR errors by creating consensus of NGS reads from the same original molecular (based on molecular barcodes and the mapping location of the reads); (3) enable the quantification of RNA by combining two types of barcodes (RNA molecular barcodes+DNA molecular barcodes), and the quantification of cfDNA (only using DNA molecular barcodes) at the same time. Because the highly expressed genes need much higher barcode diversity to identify all unique RNA fragments than cfDNA, the two-layer barcode scheme combined with Gene RADAR analysis tool enables simultaneous detection of RNA and DNA genetic alterations from a single sample.

Problems solved by technology

However, the separation of RNA from DNA can cause material loss for both DNA and RNA, and the separation process is also cost and time consuming.

Method used

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  • Systems and methods for combined detection of genetic alterations
  • Systems and methods for combined detection of genetic alterations
  • Systems and methods for combined detection of genetic alterations

Examples

Experimental program
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Effect test

example 1

llele Frequency Before and after Barcode Consensus Noise Suppression

[0065]To compare the performance of Gene RADAR and traditional next-generation sequencing using Picard deduplication, which lacks a consensus calling feature, 1.25 ng Horizon reference HD701 was spiked in to normal cfDNA to get 30 ng mixed DNA as input for PrediSeq-Pan Cancer assay. Results from Gene RADAR's analysis pipeline and traditional method were compared and are shown in FIG. 2. Using the traditional method, there were 50,820 identified variants (3,838 variants with variant frequency >0.1%). After applying Gene RADAR's consensus error correction, only 1,104 variants were identified (642 variants with variant frequency >0.1%). This indicates that Gene RADAR's consensus error correction reduces background noise by 97.8% (or 85.1% for variants with AF>0.1%). It was demonstrated that Gene RADAR's error suppression feature enables ultra-high quality sequencing of each input molecule of cfDNA.

example 2

ection Analysis Sensitivity and Accuracy

[0066]To evaluate analytic sensitivity of the PrediSeq-Pan Cancer assay, spike-in of Horizon reference DNA (HD701) was used, and 6 different SNVs were chosen for analysis. Four serial dilution ratios at 8.3%, 4.2%, 2.1%, 1.0% were used in triplicate samples to make a total 72 SNV targets. The SNV allele frequency at which >90% (18 of 20) of SNVs are detected is defined as the limit of detection, and it was calculated at 0.1% based on the data (FIG. 3). To assess the analytical accuracy of the assay's SNV AF detection, we analyzed this same set of data, choosing the SNVs with detected AF>0.1% and calculated the correlation between detected and expected AF, at 0.938.

example 3

Combined Detection has Better Coverage than cfDNA Detection Alone and Additional Sensitivity and Accuracy Contributed by cfRNA Utilization

[0067]Gene RADAR technology utilizes cfDNA and cfRNA from the same sample simultaneously, which adds additional sequencing coverage compared to cfDNA sequencing alone. For the proof of concept study, prostate cancer DNA and RNA was extracted and sequenced from Vcap cell line supernatant. The coverage differences of selected cancer related target genes in DNA only vs. DNA+RNA are presented in FIG. 3. The black dots represent the genes which have matched DNA and RNA level variants. Roughly 17.5% of genes in the selected panel benefits from at least 10% coverage, and thus higher sensitivity contributed by the cfRNA input. In additional, 46.9% of the mutations detected from cfDNA were also detected in the same location from cfRNA (supported by at least 3 reads with target mutation). Therefore, by combining DNA and RNA, additional read coverage and hig...

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Abstract

Disclosed are systems and methods for simultaneous detection of DNA and RNA genetic alterations comprising gene splicing variants, mutations, indel, copy number changes, fusion and combination thereof, in a biofluid sample from the patient without physically separating RNA from DNA. The systems and methods are similarly applicable to the simultaneous detection of DNA and RNA genetic alterations in solid tissues comprising gene splicing variants, mutations, indel, copy number changes, fusion and combination thereof. The present method utilized a barcoding method for analysis. The streamlined methods improve the simplicity, quantification accuracy and detection sensitivity and specificity of non-invasive detections of biomarkers.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS:[0001]This application is a National Stage Entry application, filed under 35 U.S.C. § 371, of International Patent Application No. PCT / US2017 / 052832, filed Sep. 21, 2017, which claims the benefit of U.S. Provisional Patent Application No. 62 / 397,923, filed Sep. 21, 2016; International Patent Application No. PCT / US2017 / 052832 is a continuation-in-part of International Patent Application No. PCT / US2017 / 027830, filed Apr. 16, 2017, each of which is incorporated by reference herein in its entirety.FIELD OF INVENTION[0002]The invention relates generally to the field of precision medicine, specifically cancer prediction, diagnostics or prognostics, and, more specifically of Gene RADAR (RNA and DNA digital Reading) and NGS methods for detecting cancer mutations in a cancer patient by the simultaneous detection of genetic alterations including RNA splicing variants, DNA- and / or RNA-based mutation, indel, long deletions, copy number variation, gene fus...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12P19/34C12Q1/6806
CPCC12Q1/6806C12Q2600/112C12Q2600/158A61K48/00C12Q1/6886C12N15/1096C40B40/06C12Q2563/179C12Q2521/107C12Q2525/161C12Q2535/122C12Q2565/514
Inventor WANG, XIAOHONGDU, PANJIA, SHIDONG
Owner PREDICINE INC
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